Indian Patents. 269010:"METHOD OF SURFACE LABELLING OF PARTICULATE SUBSTRATE"

Title of Invention	"METHOD OF SURFACE LABELLING OF PARTICULATE SUBSTRATE"
Abstract	The present invention provides a method of surface labeling of solid substrate wherein the substrate is particulate and immiscible in the solvent. The surface labeled solid substrate is useful for analytical, diagnostic and therapeutic purposes such as tracing or imaging the movement, dynamics or the physico-chemical behavior of particulate drugs or drug-delivery systcms.

Full Text	FIELD OF INVENTION The present invention relates Io a method of surface labeling of a solid substrate that involves labeling trie solid substrate that is insoluble in solvcnts containing ligand/ labei and suitable facilitating agent(s). BACKGROUND OF THE INVENTION There are several ligand-substrate interactions known to the prior art. The ligand-substrate interactions known involve liquid medium where both the ligand and the substrate are soluble in reaction solvent/ liquid medium. A facilitator that acts as reducing or catalyzing agent (s) to iniţiate or enhance the rate of binding of the ligand and the substrate is/ are also soluble in the reaction solvent / liquid medium. The liquid medium offers best opportunity for the ligand and substrate to interact at the molecular level to produce another molecule or complex, This is because of Brownian movement in the solution form that results in quick reaction at negligible time bringing closc the molecules and facilitating the interaction or labeling. The liquid phase labeling also provides a large surface area for interaction. The reaction time in case of liquid phasc labeling often ranges from nano-seconds to a maximum of a few minutes. The product of this interaction may be soluble or may precipitate as an insoluble partide or a suspended colloid. The most common medium used is water for hydrophilic compounds; organic polar solvents and hydrocarbons for hydrophobic compounds. In all the cases however, the labeled product is molecular in size. The final product can be insoluble or particulate in nature but the ratios of the substrate and ligand are molar, evenly and uniformly distributed within the solid phase. Most of inorganic or organic chemical interactions of molecular substrates and ligands făli in this category, including those commonly employed for industrial or analytical applications. SUMMARY OF THE INVENTION The present invention relates to a method of surface labeling of a solid substrate that involves labeling the solid substrate that is insoluble in solvents containing ligand/ labei and suitable facilitating agent(s). One aspect of the present disclosure relates a method of surface labeling of solid substrate, said method comprising dissolving ligand and facilitating agent in a suitable solvent; adding the substrate to the solvent to form a reaction mixture, wherein the substrate is insoluble in the solvent; stirring the reaction mixture under suitable incubation condition to form substrate ligand complex, drying the substrate ligand complex to obtain labeled substrate. Another aspect of the present disclosure relates to a kit for surface labeling of solid substrate, comprising at least one suitable solvent, ligand, facilitating agent as, instruction sheet describing surface labeling of the substrate to produce surface labeled substrate. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of surface labeling of a solid substrate that involves labeling the solid substrate that is insoluble in solvents containing ligand/ labei and suitable facilitating agent(s). One embodiment of the present disclosure relates to a method of surface labeling of a solid substrate, said method comprising: a) dissolving a ligand and a facilitating agent in a suitable solvent; b) adding the substrate to the solvent to form a reaction mixture, wherein the substrate is insoluble in the solvent; c) stirring the reaction mixture under suitable incubation condition Io form substrate ligand complex; d) drying the substrate ligand complex to obtain labeled substrate. Another embodiment of the present disclosure provides the substrate selectcd from a group consisting of salbutamol, fluticazone. chitosan, peptides, prussian blue, OPTA, GHA, MDP, BFCAs. Yet another embodiment of the present disclosure relates to the partide size of the substrate that is in micrometer or nanometer size. The method as claimed in claim l, wherein the solvent is selected from a group consisting of acetonitrile, dichloromethane, ethyl acetate, methyl ethyl ketone, ethanol, methanol, distilled water, saline, light petroleum and diethyl ether. Still another embodiment of the present disclosure provides ligand selected from a group consisting of radiolabel, fluorescent labei, magnetopharmaceutical labei and electromagnetic ray emitter. Still yct another embodiment of the present disclosure provides the facilitating agent selected from a group consisting of chelating agent, reducing agent. chloramine-T, an antibody, a bi-functional agent, and combinations thereof. The present disclosure also relates to the radiolabel selected from a group consisting of Technetium-99m, 1-131, Thallium 201, Ho and Sm. Further, the present disclosure provides the electromagnetic ray emitter such as either beta- or alpha-emitters and gamma emitters. The present disclosure provides the reducing agent such as stannous chloride and stannous tartarate. Further embodiment of the present disclosure provides the radiolabel concentration ranging from about ImCi- 20mCi. Yet another embodiment of the present disclosure provides the reducing agent concentration ranging from about 75-200mg. Still yet another embodiment of the present disclosure provides the suitable incubation condition selected from a group consisting of heating, sonication, electric current (DC) and combination thereof. The present invcntion also provides a kit for surface labeling of solid substrate. comprising at least one suitable solvent, ligand, facilitating agent and instruction sheet describing method of surface labeling of the substrate to produce surface labeled substrate. The present invention relates to solid phase labeling technique which involves suspending the insoluble particulate substrate in an immiscible fluid in a nano- or micro-particulate form. The substrate is made to react with molecular or nano or microsized ligands which are soluble/insoluble. The substrate-ligand complexes obtained from the above interaction involves attachment of the ligand either chemically or physically to the surface of the particulate substrate. This substrate-ligand complex is then used in diagnostic or therapeutic purposcs. This application of the substrate-ligand complex obtained from the invention is unique and is not possible by the usc of labeling systems known to the art. The invention is uscful in tracing or imaging movement, dynamics, physical and chemical behavior of such complexes in the in vitro environment and in-vivo environments. The term solid substrate" or substrate refers to colloid or aggregate or granules or particulate substrate and these terms have been interchangeable used herein after. The invention provides a technology to characterize the active surface properties of adsorbing particles like activated charcoal, kaoline, Prussian Blue etc. Briefly, transfer oi' a suitable radiolabel or any olher labei from the solution phase to the surface of the partide under standardized conditions will be proporţional to the amount of active surface area available for interaction. The present invention provides the formulation that is cost-effective and is useful in a number of diseases and conditions outlined above but not limited to these. The present invention relates to a solid phase labeling technique that can be used in field of diagnostics, particularly in vitro diagnostics and more particularly involving labeling of particulate or insoluble materials, with radiolabels or electromagnetic energy cmitting or absorbing tracers, using means such as chemical binding, chelation, immunc mechanisms or simply by absorption methodology. The invention is herein after referred to as solid-phase labeling or solid substratc labeling or non-solution phase labeling. This is in contrast to and is materially different from the labeling of materials in solution using the tracers or labels as is usually practiced. In contrast to the liquid phase labeling where the labei complex is uniformly present in the medium, in solid-phase labeling, the labeled complex forms a part of the surfaces of the particulate material, without substanţial changes its properties. The invention, henceforth called solid-phase labeling, is useful in diagnosis and quantification of a number of insoluble or particulate materials having an affinity for a particular labei or tracer. The invention is also useful in tracing or imaging the movement, dynamics or the physico-chemical behavior of such complexes in in vitro environment and in vivo environments, as will be clcar from the dctails mentioned below. Yet another aspect of the invention relates to the uses labeling technique for defining the surface property of the insoluble or particulate material semi-quantitatively. After fulfilling certain requirements and quality control criteria, the invention as a diagnostic tool will be able to assist the scientists and biologists in understanding the biological and physiological behavior of these substances and assists the medical professionals in diagnosis of certain diseases and estimating the pharmacological or toxic effect of these particulate substances (which can be drugs), and in tracing the pathway of particulate drugs or drug-delivery systems, particularly inside the living body. The concept of solid-phase labeling is different too, and consequently has different use, than the convenţional and routinely used liquid-phase labeling. The basic concept behind the solid state labeling or solid substrate labeling is that the substrate is particulate and immiscible in the solvent, the ligand (labei, particularly radiolabel) and the facilitating molecules (chelators/ bifunctional molecules etc.) can be either soluble or of minute size so as to interact sufficiently with the surface of substrate in the incubation period. In liquid phase labeling, thc ligand and the substrate/pharmaccutical are both soluble, and react with cach other in liquid phase (in the solvent) at molecular level. Consequently, the reaction rate is usually very fast (microseconds to a few minutes), and the rcsultant product, label-chelate-pharmaceutical or substrate complex remains either soluble (such as a radiopharmaceuitcal or a fluorescent drug) or it precipitates (like in radio-immuno-assay) and has well known applications such as in scintigraphy, RIA and various spectroscopie analysis variants. The guiding principie is the uniformity of dispersion within the solvent and the chelation/absorption/chemical reaction too is totally uniform and has no spaţial distinctions. In solid-phase labeling on the other hand, the substrate is a crystal/granule/ colloid/particle/aggregate which is suspended in the solvent whcre essentially it is insoluble. l'he substrate ideally rctains its physical shapc and dimensions within thc fluid system and rctains its essential characteristics even in thc dried form. The substrate is suspended in the immiscible liquid containing the ligand with which it has affinity (the affinity needs to be confirmed in prior liquid phase interaction) and other facilitating chemicals. The ligand interacts which the substrate molecules present on the particulate surface as thcse are the only ones it is in contact with. The reaction is expected to proceed at a slow rate but reaches equilibrium similar to the convenţional reaction. The liquid phase is then either dried out, or the particulate substrate filtered out for applications. AII the applications are centered around the fact that the surface of the substrate is now labeled with the ligand and have funcţional properties of the ligand while maintaining the structural integrity of the substrate. One of the main advantages is that the interiors of the particulate are not labeled by the ligand and the interior is capable of serving its natural useful purpose as the ligand shall only be confined to the surface of the particulate substrate and the structure of the particulate substrate is not irreversibly lost in the process. The solid-phase labeling assembly can be made available in the form of a kit with a minimum of two preferred components. The first component is a single or a combination of labels or ligand, whether fluorescent, radioactive (particularly based on technctium-99 or FDG chemistry), magnctopharmaccutical. or emitter or absorber of any other part of electromagnetic spectrum. The second preferred component is a chelator, a reducer, an antibody, a bi-functional agent or any chemical(s) or a substance(s) that has the ability to bind or affix the labei or the tracer to the surface of the particulate substrate to be labeled. The nomenclature and the quantity of the kit material are variable and are dependent on use. The present invention further relates to the derivation of the colloidal phase labeling or interaction resulting in surface labeled molecules, substances, technologies and applications. The present invention involves surface labeling of a colloid / aggregate / granules or such particulate substrates suspended in an immiscible solvent with a labei. The biophysical properties of the substrate after formation of the substrate-ligand complex remain the same. The present invention is expected to fulfill a number of necds of the industry, analytical units and human diagnostics and therapeutics. Few representative examples are given below. However, the list so presented is not complete and does not preclude the inventor's right to usc it in other situations not mentioned hereupon. In radionuclide imaging of the human respiratory tree using radiolabelled micronised drugs, there is a need to image the distribution of the drug upon inhalation (by dry powder inhalation) because the distribution of the drug predominantly defines the efficacy of inhalation therapy in that particular person. This inibrmation is needed most in case of patients with asthma, bronchitis, COPD, and various other obstructive and restrictive diseases of the respiratory system and is presently provided by radio-adsorption which is an imperfect technique and is not fully correct or objective. In radionuclide imaging of lung ventilation, there is a need for lung ventilation imaging for diagnostic purposes. Presently, it is done by Xenon-113 and wet nubuli/ation method. Both are cumbersome, wasteful and lead to environmental pollution with radioactivity. In imaging in vivo dynamics of radio labeled particulate drugs, scintigraphy is used as the gold standard for tracing the pathway of radiolabeled drugs in the human or animal body. However, the labeling is at molecular level. Most of the drug formulations, particularly oral drugs, are given in particulate form. There is a need for surlace labeling of particulate forms, which will open a new avenue for research related applications. In radiotherapy with particulate material the nuclear medicine therapy is done largely by molecular (radio-iodine, P-32), macro-molecular (alpha-, beta emitters) or colloids (Holonium-HHDP colloid), where radioactivity is chemically attached to the substrate. A problem with non-particulate material is that these are broken down quite early in the body thus limiting the efficacy of the radiotherapy and increasing body burden of the radiation. Labeling of substrate in an immiscible medium, particularly surface labeling of particulates that are not usually dissolved in body fluids is needed for research related applications. The present invention that relatcs to method of solid substrate labeling is particularly helpful as an analytical tool as at present there is no method for assessing the reactive surface area of surface active particulate materials like activated charcoal, silica gel & Prussian Blue. The present invention relates to method of solid substrate labeling which is helpful as at present there is no method for chemical labeling of pre-formed particles for therapeutic or diagnostic purposes. The present invention to method of solid substrate labeling where the surface labeled solid substrate can be used in cancer detection by use of labeled particulate material in techniques such as bronchography or endoscopy where the labeled particulate material is liberated in the vicinity of cancerous cells, example the cancer cells of the lung or other ENT or proximal GIT cancers. Example l relates to a method of radiolabeling the surface of a particulate drug, particularly a beta agonist drug or a steroid, preferably salbutamol sulphatc in microni/ed tbrm, with Technetium-99m. These products are to be used for imaging the distribution of the drug in the human respiratory tree which has diagnostic value. Example 2 relates to a method of radiolabel the surface of a particulate drug, preferably steroids and smooth muscle relaxants or beta agonist drug, preferably salbutamol in nanometer size range, with Technetium-99m. These products are to be used for imaging the distribution of the drug in alveoli which has diagnostic value. Further, the method of radiolabeling the surface of an excipient, preferably lactose in nanometer size range, with Technetium-99m is provided. These products are for imaging the distribution of the drug in alveoli, and have diagnostic value for ventilation lung scanning as dry powder inhaler. Example 4 relates to a method of radiolabeling the surface of a particulate drug, DTPA with Technetium-99m. This radiolabelled complex is further milled into products such as like tabiet, capsule and spansule to be administered to humans. The study is to be used for imaging the distribution of the drug in gastrointestinal tract or in any other system of the body. Kxamplc 5 relates to a method of radiolabeling the surface of a particulate drug, MDP, with Technetium-99m. This radiolabelled complex is further milled into products such as like tabiet, capsule and spansule to be administered to humans. The study is to be used for imaging the distribution of the drug in gastrointestinal tract or in any other system of the body. Example 6 relates to a method of radiolabeling the surface of a particulate drug, salbutamol sulphate, amenable for surface labeling for therapeutic or diagnostic purposes. While the present invention has been described in terms of specific methods and compositions, it is understood that variations and modifications will occur to those skilled in the art upon consideration of the present invention. The ibrmulations and products are described as embodiments of the present invention but are to same and have no equivalent counterparts in the present day technology. EXAMPLES It should be understood that the following examples described herein are only for illustrative purposes and all the possible modifications or changes suggested to a person skilled in the art are included within the scope and purview of this application and appended claims. EXAMPLE l 200 micrograms of micronized salbutamol sulphate is suspended in an adequate quantity of organic solvent like di-ethyl ether. A soluble form of Tc-99m (lOmCi) is made to react with the surface of the particulate salbutamol upon incubation in preferred conditions such as agitation by heating and sonification. Reducing agents like stannous ions (150 micrograms) can be introduced in the milieu beforehand to facilitate chemical chelation of the ligand (Tc-99m) with the surface molcculcs of the particulate drug. The complex is then dried by cvaporating the vehicle medium to give powdered form of microni/ed radiolabeled Tc-99m salbutamol. The labeled particulate product is then mixed with medicated lactose powder through convenţional methods, and inhaled as in Dry Powder Inhalation (DPI) which acts as a therapeutic- diagnostic product. Gamma Camera imaging is done at intervals to acquire important diagnostic information and predicting therapeutic benefits of the drug treatment for individual treatments. The quality control assessment was done by Instant thin-layer chromatography (ITLC) to confirm chelation. EXAMPLE 2 200 micrograms of salbutamol sulphate or lactose nanoparticles were made and suspended in an adequate quantity of organic solvent like acetone. A soluble form of Tc-99m (10 mCi) is made to react with the surface of the particulate lactose upon incubation in preferred conditions such as agitation by heating, & sonification etc. A reducing agent, stannous chloride (40 micrograms) was pre-introduced in the milieu to facilitate the chemical chelation of the ligand (Tc-99m) with the surface molecules of the particulate drug (substrate). The complex was then dried by evaporating the vehicle medium to give powdered form of micronized Radiolabeled Tc-99m nano-lactose. Incubation of 15 minutes is essential for room temperature conditions. The labeled particulate product is then mixed with medicated lactose powder through convenţional methods, and inhaled as Dry Powder Inhalation (DPI). Gamma Camera imaging is done at intervals to acquire important diagnostic information and predicting therapeutic benefits of the drug treatment for individual treatments. Biodistribution of Tc-99m labeled nanoparticles of salbutamol give information relevant to the therapeutic planning of the individual. Tc-99m labeled nanoparticles of lactose scan is used as ventilation lung scans for disease characterization. Quality control was done by ITLC to confirm the chclation. EXAMPLK 3 500 microgram of L)TPA particles were suspended in acetonitrile already containing 2 mCi of Tc-99m pertechnetate and 80 microgram of stannous tartarate. After incubation for l hr in standard conditions, the particles were dried off and mixed in dried form with 499.5 mg of parent DTPA powder. A sustained release formulation was made from the mixture using standard protocols. Release rates of the radiolabel were confirmed to be equivalent to the release rate of cold DPT A in the particulate form through standard in vitro analytical methods. The formulation was then given to a human being and important information gained regarding the release rates of the formulation. EXAMPLE 4 500 microgram of MDP particles were suspended in P-11 (a commercial solvent approvcd for inhalation) containing 2 mCi of Tc-99m pertechnetate and 80 microgram of stannous tartrate. After incubation for l hr in standard conditions, the particles were dried off and mixed in dried form with 19.5 mg of parent MDP powder. The mixture was re-suspended in P-11 solvent and packed in pressuri/ed canister using a standard technique. The Metered Dose Inhaler (MDI of Tc-MDP particulate) was inhaled by volunteer to give biodistribution of the drug in the lungs. The methodology, as described above, served us to develop a new product (phosphonate MDI) that may have use in allergic respiratory conditions and as a respiratory chelating agent. EXAMPLE 5 200 micrograms of micronized salbutamol sulphate is suspended in an adequate quantity of methyl ethyl ketone. A soluble form of radioiodine (l mCi) was made to react with the surface of the particulate salbutamol upon incubation in preferred conditions such as agitation by heating and sonification and convenţional methods likc chloramine-T mcthod. The complex is thcn dried by evaporating the vehicle mcdium to obtain powercd form of microni/cd Radioiodinated salbutamol as a therapeutic agent. In the above experiments it was observed that the surface labeled solid substrate has stability at about pH 7.4 or other specified environmental conditions. The surface labeled solid substrate showed no change in the partide size, charge and other particulate characteristic. Further, labeling of more than 70% was achieved. The surface labeled solid substrate hâd biological life similar to the physical half life and the substrate hâd no acquired toxicity. The surface labeled solid substrate can be used for several purposes such as in vivo imaging as well as therapeutics. There was no lethal radiation within a range of 1-2 cm with no other preferred site of deposition except the intended target. lAVe claim: 1. A method of surface labcling of a solid substrate, said method comprising: e) dissolving a ligand and a facilitating agent in a suitable solvent; O adding the substrate to the solvent to form a reaction mixture, wherein the substrate is insoluble in the solvent; g) stirring the reaction mixture under suitable incubation condition to form substrate ligand complex; h) drying the substrate ligand complex to obtain labeled substrate. 2. The method as claimed in claim l, wherein the substrate is selected from a group consisting of salbutamol, fluticazone, chitosan, peptides, prussian blue, DPTA, GHA, MDP, BFCAs. 3. The method as claimed in claim l, wherein the substrate is in micrometer or nanometer si/e range. 4. The method as claimed in claim l, wherein the solvent is selected from a group consisting of acetonitrile, dichloromethane, ethyl acetate, methyl ethyl ketone, ethanol, methanol, distilled water, saline, light petroleum and diethyl ether. 5. The method as claimed in claim l, wherein the ligand is selected from a group consisting of radiolabel, fluorescent labei, magnetopharmaceutical labei and electromagnetic ray emitter. 6. The method as claimed in claim l, wherein the facilitating agent is selected from a group consisting of chelating agent, reducing agent, chloramine-T, an antibody, a bi-functional agent, and combinations thereof. 7. The method as claimed in claim 5, wherein the radiolabel is selected from a group consisting of Technetium-99m, 1-131, Thallium 201, Ho and Sm. 8. The method as claimed in claim 5, wherein the electromagnetic ray emitter is either beta- or alpha-emitters or gamma emitters. 9. The method as claimed in claim 6, wherein the reducing agent is either stannous chloride or stannous tartarate. 10. The method as claimed in claim 5, wherein the radiolabel concentration ranges from about ImCi- 20mCi. 11. The method as claimed in claim 6, wherein the reducing agent concentration ranges from about 75-200mg. 12. The method as claimed in claim l, wherein the suitable incubation condition is selected from a group consisting of heating, sonication, electric current (DC) and combination thereof. 13. A kit for surface labeling of solid substrate, comprising at least one suitable solvent as claimed in claim 4, ligand as claimed in claim 5, facilitating agent as claimed in claim 6 and instruction sheet describing method of surface labeling of the substrate to produce surface labeled substrate.

Full Text

FIELD OF INVENTION
The present invention relates Io a method of surface labeling of a solid substrate that involves labeling trie solid substrate that is insoluble in solvcnts containing ligand/ labei and suitable facilitating agent(s).
BACKGROUND OF THE INVENTION
There are several ligand-substrate interactions known to the prior art. The ligand-substrate interactions known involve liquid medium where both the ligand and the substrate are soluble in reaction solvent/ liquid medium. A facilitator that acts as reducing or catalyzing agent (s) to iniţiate or enhance the rate of binding of the ligand and the substrate is/ are also soluble in the reaction solvent / liquid medium. The liquid medium offers best opportunity for the ligand and substrate to interact at the molecular level to produce another molecule or complex, This is because of Brownian movement in the solution form that results in quick reaction at negligible time bringing closc the molecules and facilitating the interaction or labeling. The liquid phase labeling also provides a large surface area for interaction. The reaction time in case of liquid phasc labeling often ranges from nano-seconds to a maximum of a few minutes. The product of this interaction may be soluble or may precipitate as an insoluble partide or a suspended colloid. The most common medium used is water for hydrophilic compounds; organic polar solvents and hydrocarbons for hydrophobic compounds. In all the cases however, the labeled product is molecular in size. The final product can be insoluble or particulate in nature but the ratios of the substrate and ligand are molar, evenly and uniformly distributed within the solid phase. Most of inorganic or organic chemical interactions of molecular substrates and ligands făli in this category, including those commonly employed for industrial or analytical applications.
SUMMARY OF THE INVENTION
The present invention relates to a method of surface labeling of a solid substrate that involves labeling the solid substrate that is insoluble in solvents containing ligand/ labei and suitable facilitating agent(s).
One aspect of the present disclosure relates a method of surface labeling of solid substrate, said method comprising dissolving ligand and facilitating agent in a suitable solvent; adding the substrate to the solvent to form a reaction mixture, wherein the substrate is insoluble in the solvent; stirring the reaction mixture under suitable incubation condition to form substrate ligand complex, drying the substrate ligand complex to obtain labeled substrate.
Another aspect of the present disclosure relates to a kit for surface labeling of solid substrate, comprising at least one suitable solvent, ligand, facilitating agent as, instruction sheet describing surface labeling of the substrate to produce surface labeled substrate.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of surface labeling of a solid substrate that involves labeling the solid substrate that is insoluble in solvents containing ligand/ labei and suitable facilitating agent(s).
One embodiment of the present disclosure relates to a method of surface labeling of a solid substrate, said method comprising:
a) dissolving a ligand and a facilitating agent in a suitable solvent;
b) adding the substrate to the solvent to form a reaction mixture, wherein the
substrate is insoluble in the solvent;
c) stirring the reaction mixture under suitable incubation condition Io form
substrate ligand complex;
d) drying the substrate ligand complex to obtain labeled substrate.
Another embodiment of the present disclosure provides the substrate selectcd from a group consisting of salbutamol, fluticazone. chitosan, peptides, prussian blue, OPTA, GHA, MDP, BFCAs.
Yet another embodiment of the present disclosure relates to the partide size of the substrate that is in micrometer or nanometer size.
The method as claimed in claim l, wherein the solvent is selected from a group consisting of acetonitrile, dichloromethane, ethyl acetate, methyl ethyl ketone, ethanol, methanol, distilled water, saline, light petroleum and diethyl ether.
Still another embodiment of the present disclosure provides ligand selected from a group consisting of radiolabel, fluorescent labei, magnetopharmaceutical labei and electromagnetic ray emitter.
Still yct another embodiment of the present disclosure provides the facilitating agent selected from a group consisting of chelating agent, reducing agent. chloramine-T, an antibody, a bi-functional agent, and combinations thereof.
The present disclosure also relates to the radiolabel selected from a group consisting of Technetium-99m, 1-131, Thallium 201, Ho and Sm.
Further, the present disclosure provides the electromagnetic ray emitter such as either beta- or alpha-emitters and gamma emitters.
The present disclosure provides the reducing agent such as stannous chloride and stannous tartarate.
Further embodiment of the present disclosure provides the radiolabel concentration ranging from about ImCi- 20mCi.
Yet another embodiment of the present disclosure provides the reducing agent concentration ranging from about 75-200mg.
Still yet another embodiment of the present disclosure provides the suitable incubation condition selected from a group consisting of heating, sonication, electric current (DC) and combination thereof.
The present invcntion also provides a kit for surface labeling of solid substrate. comprising at least one suitable solvent, ligand, facilitating agent and instruction sheet describing method of surface labeling of the substrate to produce surface labeled substrate.
The present invention relates to solid phase labeling technique which involves suspending the insoluble particulate substrate in an immiscible fluid in a nano- or micro-particulate form. The substrate is made to react with molecular or nano or microsized ligands which are soluble/insoluble. The substrate-ligand complexes obtained from the above interaction involves attachment of the ligand either chemically or physically to the surface of the particulate substrate. This substrate-ligand complex is then used in diagnostic or therapeutic purposcs. This application of the substrate-ligand complex obtained from the invention is unique and is not possible by the usc of labeling systems known to the art. The invention is uscful in tracing or imaging movement, dynamics, physical and chemical behavior of such complexes in the in vitro environment and in-vivo environments.
The term solid substrate" or substrate refers to colloid or aggregate or granules or particulate substrate and these terms have been interchangeable used herein after.
The invention provides a technology to characterize the active surface properties of adsorbing particles like activated charcoal, kaoline, Prussian Blue etc. Briefly,
transfer oi' a suitable radiolabel or any olher labei from the solution phase to the surface of the partide under standardized conditions will be proporţional to the amount of active surface area available for interaction.
The present invention provides the formulation that is cost-effective and is useful in a number of diseases and conditions outlined above but not limited to these.
The present invention relates to a solid phase labeling technique that can be used in field of diagnostics, particularly in vitro diagnostics and more particularly involving labeling of particulate or insoluble materials, with radiolabels or electromagnetic energy cmitting or absorbing tracers, using means such as chemical binding, chelation, immunc mechanisms or simply by absorption methodology. The invention is herein after referred to as solid-phase labeling or solid substratc labeling or non-solution phase labeling. This is in contrast to and is materially different from the labeling of materials in solution using the tracers or labels as is usually practiced. In contrast to the liquid phase labeling where the labei complex is uniformly present in the medium, in solid-phase labeling, the labeled complex forms a part of the surfaces of the particulate material, without substanţial changes its properties.
The invention, henceforth called solid-phase labeling, is useful in diagnosis and quantification of a number of insoluble or particulate materials having an affinity for a particular labei or tracer. The invention is also useful in tracing or imaging the movement, dynamics or the physico-chemical behavior of such complexes in in vitro environment and in vivo environments, as will be clcar from the dctails mentioned below.
Yet another aspect of the invention relates to the uses labeling technique for defining the surface property of the insoluble or particulate material semi-quantitatively. After fulfilling certain requirements and quality control criteria, the invention as a diagnostic tool will be able to assist the scientists and biologists in understanding the biological and physiological behavior of these substances and
assists the medical professionals in diagnosis of certain diseases and estimating the pharmacological or toxic effect of these particulate substances (which can be drugs), and in tracing the pathway of particulate drugs or drug-delivery systems, particularly inside the living body.
The concept of solid-phase labeling is different too, and consequently has different use, than the convenţional and routinely used liquid-phase labeling. The basic concept behind the solid state labeling or solid substrate labeling is that the substrate is particulate and immiscible in the solvent, the ligand (labei, particularly radiolabel) and the facilitating molecules (chelators/ bifunctional molecules etc.) can be either soluble or of minute size so as to interact sufficiently with the surface of substrate in the incubation period.
In liquid phase labeling, thc ligand and the substrate/pharmaccutical are both soluble, and react with cach other in liquid phase (in the solvent) at molecular level. Consequently, the reaction rate is usually very fast (microseconds to a few minutes), and the rcsultant product, label-chelate-pharmaceutical or substrate complex remains either soluble (such as a radiopharmaceuitcal or a fluorescent drug) or it precipitates (like in radio-immuno-assay) and has well known applications such as in scintigraphy, RIA and various spectroscopie analysis variants. The guiding principie is the uniformity of dispersion within the solvent and the chelation/absorption/chemical reaction too is totally uniform and has no spaţial distinctions.
In solid-phase labeling on the other hand, the substrate is a crystal/granule/ colloid/particle/aggregate which is suspended in the solvent whcre essentially it is insoluble. l'he substrate ideally rctains its physical shapc and dimensions within thc fluid system and rctains its essential characteristics even in thc dried form.
The substrate is suspended in the immiscible liquid containing the ligand with which it has affinity (the affinity needs to be confirmed in prior liquid phase interaction) and other facilitating chemicals. The ligand interacts which the
substrate molecules present on the particulate surface as thcse are the only ones it is in contact with. The reaction is expected to proceed at a slow rate but reaches equilibrium similar to the convenţional reaction. The liquid phase is then either dried out, or the particulate substrate filtered out for applications. AII the applications are centered around the fact that the surface of the substrate is now labeled with the ligand and have funcţional properties of the ligand while maintaining the structural integrity of the substrate. One of the main advantages is that the interiors of the particulate are not labeled by the ligand and the interior is capable of serving its natural useful purpose as the ligand shall only be confined to the surface of the particulate substrate and the structure of the particulate substrate is not irreversibly lost in the process.
The solid-phase labeling assembly can be made available in the form of a kit with a minimum of two preferred components. The first component is a single or a combination of labels or ligand, whether fluorescent, radioactive (particularly based on technctium-99 or FDG chemistry), magnctopharmaccutical. or emitter or absorber of any other part of electromagnetic spectrum. The second preferred component is a chelator, a reducer, an antibody, a bi-functional agent or any chemical(s) or a substance(s) that has the ability to bind or affix the labei or the tracer to the surface of the particulate substrate to be labeled. The nomenclature and the quantity of the kit material are variable and are dependent on use.
The present invention further relates to the derivation of the colloidal phase labeling or interaction resulting in surface labeled molecules, substances, technologies and applications. The present invention involves surface labeling of a colloid / aggregate / granules or such particulate substrates suspended in an immiscible solvent with a labei. The biophysical properties of the substrate after formation of the substrate-ligand complex remain the same.
The present invention is expected to fulfill a number of necds of the industry, analytical units and human diagnostics and therapeutics. Few representative
examples are given below. However, the list so presented is not complete and does not preclude the inventor's right to usc it in other situations not mentioned hereupon.
In radionuclide imaging of the human respiratory tree using radiolabelled micronised drugs, there is a need to image the distribution of the drug upon inhalation (by dry powder inhalation) because the distribution of the drug predominantly defines the efficacy of inhalation therapy in that particular person. This inibrmation is needed most in case of patients with asthma, bronchitis, COPD, and various other obstructive and restrictive diseases of the respiratory system and is presently provided by radio-adsorption which is an imperfect technique and is not fully correct or objective.
In radionuclide imaging of lung ventilation, there is a need for lung ventilation imaging for diagnostic purposes. Presently, it is done by Xenon-113 and wet nubuli/ation method. Both are cumbersome, wasteful and lead to environmental pollution with radioactivity.
In imaging in vivo dynamics of radio labeled particulate drugs, scintigraphy is used as the gold standard for tracing the pathway of radiolabeled drugs in the human or animal body. However, the labeling is at molecular level. Most of the drug formulations, particularly oral drugs, are given in particulate form. There is a need for surlace labeling of particulate forms, which will open a new avenue for research related applications.
In radiotherapy with particulate material the nuclear medicine therapy is done largely by molecular (radio-iodine, P-32), macro-molecular (alpha-, beta emitters) or colloids (Holonium-HHDP colloid), where radioactivity is chemically attached to the substrate. A problem with non-particulate material is that these are broken down quite early in the body thus limiting the efficacy of the radiotherapy and increasing body burden of the radiation. Labeling of substrate in an immiscible
medium, particularly surface labeling of particulates that are not usually dissolved in body fluids is needed for research related applications.
The present invention that relatcs to method of solid substrate labeling is particularly helpful as an analytical tool as at present there is no method for assessing the reactive surface area of surface active particulate materials like activated charcoal, silica gel & Prussian Blue.
The present invention relates to method of solid substrate labeling which is helpful as at present there is no method for chemical labeling of pre-formed particles for therapeutic or diagnostic purposes.
The present invention to method of solid substrate labeling where the surface labeled solid substrate can be used in cancer detection by use of labeled particulate material in techniques such as bronchography or endoscopy where the labeled particulate material is liberated in the vicinity of cancerous cells, example the cancer cells of the lung or other ENT or proximal GIT cancers.
Example l relates to a method of radiolabeling the surface of a particulate drug, particularly a beta agonist drug or a steroid, preferably salbutamol sulphatc in microni/ed tbrm, with Technetium-99m. These products are to be used for imaging the distribution of the drug in the human respiratory tree which has diagnostic value.
Example 2 relates to a method of radiolabel the surface of a particulate drug, preferably steroids and smooth muscle relaxants or beta agonist drug, preferably salbutamol in nanometer size range, with Technetium-99m. These products are to be used for imaging the distribution of the drug in alveoli which has diagnostic value.
Further, the method of radiolabeling the surface of an excipient, preferably lactose in nanometer size range, with Technetium-99m is provided. These products are for
imaging the distribution of the drug in alveoli, and have diagnostic value for ventilation lung scanning as dry powder inhaler.
Example 4 relates to a method of radiolabeling the surface of a particulate drug, DTPA with Technetium-99m. This radiolabelled complex is further milled into products such as like tabiet, capsule and spansule to be administered to humans. The study is to be used for imaging the distribution of the drug in gastrointestinal tract or in any other system of the body.
Kxamplc 5 relates to a method of radiolabeling the surface of a particulate drug, MDP, with Technetium-99m. This radiolabelled complex is further milled into products such as like tabiet, capsule and spansule to be administered to humans. The study is to be used for imaging the distribution of the drug in gastrointestinal tract or in any other system of the body.
Example 6 relates to a method of radiolabeling the surface of a particulate drug, salbutamol sulphate, amenable for surface labeling for therapeutic or diagnostic purposes.
While the present invention has been described in terms of specific methods and compositions, it is understood that variations and modifications will occur to those skilled in the art upon consideration of the present invention.
The ibrmulations and products are described as embodiments of the present invention but are to same and have no equivalent counterparts in the present day technology.
EXAMPLES
It should be understood that the following examples described herein are only for illustrative purposes and all the possible modifications or changes suggested to a
person skilled in the art are included within the scope and purview of this application and appended claims.
EXAMPLE l
200 micrograms of micronized salbutamol sulphate is suspended in an adequate quantity of organic solvent like di-ethyl ether. A soluble form of Tc-99m (lOmCi) is made to react with the surface of the particulate salbutamol upon incubation in preferred conditions such as agitation by heating and sonification. Reducing agents like stannous ions (150 micrograms) can be introduced in the milieu beforehand to facilitate chemical chelation of the ligand (Tc-99m) with the surface molcculcs of the particulate drug. The complex is then dried by cvaporating the vehicle medium to give powdered form of microni/ed radiolabeled Tc-99m salbutamol.
The labeled particulate product is then mixed with medicated lactose powder through convenţional methods, and inhaled as in Dry Powder Inhalation (DPI) which acts as a therapeutic- diagnostic product. Gamma Camera imaging is done at intervals to acquire important diagnostic information and predicting therapeutic benefits of the drug treatment for individual treatments. The quality control assessment was done by Instant thin-layer chromatography (ITLC) to confirm chelation.
EXAMPLE 2
200 micrograms of salbutamol sulphate or lactose nanoparticles were made and suspended in an adequate quantity of organic solvent like acetone. A soluble form of Tc-99m (10 mCi) is made to react with the surface of the particulate lactose upon incubation in preferred conditions such as agitation by heating, & sonification etc. A reducing agent, stannous chloride (40 micrograms) was pre-introduced in the milieu to facilitate the chemical chelation of the ligand (Tc-99m) with the surface molecules of the particulate drug (substrate). The complex was
then dried by evaporating the vehicle medium to give powdered form of micronized Radiolabeled Tc-99m nano-lactose. Incubation of 15 minutes is essential for room temperature conditions.
The labeled particulate product is then mixed with medicated lactose powder through convenţional methods, and inhaled as Dry Powder Inhalation (DPI). Gamma Camera imaging is done at intervals to acquire important diagnostic information and predicting therapeutic benefits of the drug treatment for individual treatments. Biodistribution of Tc-99m labeled nanoparticles of salbutamol give information relevant to the therapeutic planning of the individual. Tc-99m labeled nanoparticles of lactose scan is used as ventilation lung scans for disease characterization. Quality control was done by ITLC to confirm the chclation.
EXAMPLK 3
500 microgram of L)TPA particles were suspended in acetonitrile already containing 2 mCi of Tc-99m pertechnetate and 80 microgram of stannous tartarate. After incubation for l hr in standard conditions, the particles were dried off and mixed in dried form with 499.5 mg of parent DTPA powder. A sustained release formulation was made from the mixture using standard protocols.
Release rates of the radiolabel were confirmed to be equivalent to the release rate of cold DPT A in the particulate form through standard in vitro analytical methods. The formulation was then given to a human being and important information gained regarding the release rates of the formulation.
EXAMPLE 4
500 microgram of MDP particles were suspended in P-11 (a commercial solvent approvcd for inhalation) containing 2 mCi of Tc-99m pertechnetate and 80 microgram of stannous tartrate. After incubation for l hr in standard conditions, the particles were dried off and mixed in dried form with 19.5 mg of parent MDP
powder. The mixture was re-suspended in P-11 solvent and packed in pressuri/ed canister using a standard technique.
The Metered Dose Inhaler (MDI of Tc-MDP particulate) was inhaled by volunteer to give biodistribution of the drug in the lungs. The methodology, as described above, served us to develop a new product (phosphonate MDI) that may have use in allergic respiratory conditions and as a respiratory chelating agent.
EXAMPLE 5
200 micrograms of micronized salbutamol sulphate is suspended in an adequate quantity of methyl ethyl ketone. A soluble form of radioiodine (l mCi) was made to react with the surface of the particulate salbutamol upon incubation in preferred conditions such as agitation by heating and sonification and convenţional methods likc chloramine-T mcthod. The complex is thcn dried by evaporating the vehicle mcdium to obtain powercd form of microni/cd Radioiodinated salbutamol as a therapeutic agent.
In the above experiments it was observed that the surface labeled solid substrate has stability at about pH 7.4 or other specified environmental conditions. The surface labeled solid substrate showed no change in the partide size, charge and other particulate characteristic. Further, labeling of more than 70% was achieved. The surface labeled solid substrate hâd biological life similar to the physical half life and the substrate hâd no acquired toxicity. The surface labeled solid substrate can be used for several purposes such as in vivo imaging as well as therapeutics. There was no lethal radiation within a range of 1-2 cm with no other preferred site of deposition except the intended target.

lAVe claim:
1. A method of surface labcling of a solid substrate, said method comprising:
e) dissolving a ligand and a facilitating agent in a suitable solvent;
O adding the substrate to the solvent to form a reaction mixture, wherein the substrate is insoluble in the solvent;
g) stirring the reaction mixture under suitable incubation condition to form substrate ligand complex;
h) drying the substrate ligand complex to obtain labeled substrate.
2. The method as claimed in claim l, wherein the substrate is selected from a
group consisting of salbutamol, fluticazone, chitosan, peptides, prussian blue,
DPTA, GHA, MDP, BFCAs.
3. The method as claimed in claim l, wherein the substrate is in micrometer or
nanometer si/e range.
4. The method as claimed in claim l, wherein the solvent is selected from a
group consisting of acetonitrile, dichloromethane, ethyl acetate, methyl ethyl
ketone, ethanol, methanol, distilled water, saline, light petroleum and diethyl
ether.
5. The method as claimed in claim l, wherein the ligand is selected from a
group consisting of radiolabel, fluorescent labei, magnetopharmaceutical
labei and electromagnetic ray emitter.
6. The method as claimed in claim l, wherein the facilitating agent is selected
from a group consisting of chelating agent, reducing agent, chloramine-T, an
antibody, a bi-functional agent, and combinations thereof.
7. The method as claimed in claim 5, wherein the radiolabel is selected from a
group consisting of Technetium-99m, 1-131, Thallium 201, Ho and Sm.
8. The method as claimed in claim 5, wherein the electromagnetic ray emitter is
either beta- or alpha-emitters or gamma emitters.
9. The method as claimed in claim 6, wherein the reducing agent is either
stannous chloride or stannous tartarate.
10. The method as claimed in claim 5, wherein the radiolabel concentration
ranges from about ImCi- 20mCi.
11. The method as claimed in claim 6, wherein the reducing agent concentration
ranges from about 75-200mg.
12. The method as claimed in claim l, wherein the suitable incubation condition
is selected from a group consisting of heating, sonication, electric current
(DC) and combination thereof.
13. A kit for surface labeling of solid substrate, comprising at least one suitable
solvent as claimed in claim 4, ligand as claimed in claim 5, facilitating agent
as claimed in claim 6 and instruction sheet describing method of surface
labeling of the substrate to produce surface labeled substrate.

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Patent Number

269010

Indian Patent Application Number

729/DEL/2007

PG Journal Number

40/2015

Publication Date

02-Oct-2015

Grant Date

28-Sep-2015

Date of Filing

30-Mar-2007

Name of Patentee

DIRECTOR GENERAL DEFENCE RESEARCH & DEVELOPMENT ORGANISATION

Applicant Address

MIN.OF DEFENCE,GOVT.OF INDIA,DIRECTORATE OF ER & IPR,IPR GROUP,ROOM NO.348,"DRDO BHAVAN",RAJAJI MARG, NEW DELHI-110011

Inventors:

#	Inventor's Name	Inventor's Address
1	GOOMER CHANDER NARESH	INMAS, BRIG.SK MAZUMDAR MARG DELHI-110054
2	BHATNAGAR ASEEM	INMAS, BRIG.SK MAZUMDAR MARG DELHI-110054
3	SACHDEV RUCHI	INMAS, BRIG.SK MAZUMDAR MARG DELHI-110054

PCT International Classification Number

A61K51/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA